Butyrate Ameliorates Insufficient Sleep-Induced Intestinal Mucosal Damage in Humans and Mice

ABSTRACT Insufficient sleep is a key factor in the occurrence of intestinal diseases. This study was performed to clarify how sleep deficiency mediates the intestinal microbiota, metabolite butyrate disturbance induces intestinal mucosal damage, and butyrate ameliorates it. A questionnaire was launched for sleep and intestinal health issues. Twenty-two healthy volunteers were interviewed, and the influence of insufficient sleep on the gut microbiota and metabolite composition was explored. Moreover, a 72-h sleep deprivation (SD) mouse model with or without butyrate supplementation was used to reveal the effect of butyrate on ameliorating small intestines damage caused by SD. The questionnaire survey of 534 college students showed that among 85.39% of the students who slept less than 7 h, 41.76% were suffering from various bowel disorders. High-throughput 16S rRNA pyrosequencing demonstrated that SD and sleep restriction (SR) resulted in downregulation of Faecalibacterium and butyrate abundance in the feces of college students. Furthermore, we observed that butyrate supplementation markedly reversed sleep-deprivation-induced small intestinal mucosal injury in mice. Meanwhile, butyrate supplementation inverted the SD-caused inflammation response and oxidative stress and the decline of phospho-glycogen synthase kinase 3β (p-GSK-3β), β-catenin, Nrf2, and cyclin D1 and the increase in histone deacetylase 3 (HDAC3) and phospho-P65 (p-P65) proteins in the small intestines. Furthermore, in vitro, the ameliorative effects of butyrate were blocked by treatment with the HDAC3 agonist ITSA-1 and the Nrf2 antagonist ML385 and mimicked by treatment with the HDAC3 antagonist RGFP966 and p-P65 antagonist PDTC. Our study revealed that SD and SR downregulated butyrate production, further causing intestinal homeostasis dysfunction via the HDAC3–p-GSK-3β–β-catenin–Nrf2–NF-κB pathway. IMPORTANCE Radical inflammatory bowel disease (IBD) induced by sleep deficiency is a serious global public health threat. Butyrate, a member of the short-chain fatty acids, exerts multiple effects on it. However, existing research focuses on injury to the colon caused by insufficient sleep, while the changes in the small intestines are often overlooked. This study focused on revealing the influence of insufficient sleep on the intestinal microbiota and its metabolites and further revealed the ameliorative effect of butyrate on sleep deprivation (SD)-induced small intestinal mucosal damage in human and mice. Our studies suggest that butyrate can be used as a probiotic to restore SD-induced IBD and contribute to a better understanding of the mechanisms that govern the beneficial effects of butyrate.

(PCA), principal coordinate analysis (PCoA), and nonmetric multidimensional scaling (NMDS) were performed using QIIME. LEfSe analysis was performed for the quantitative analysis of biomarkers among each group. Briefly, the LEfSe analysis, LDA threshold of > 4, used the nonparametric factorial Kruskal-Wallis (KW) sum-rank test and then the (unpaired) Wilcoxon rank-sum test to identify the most differently abundant taxa. The difference in dominant pathways between groups was detected using Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and clusters of orthologous groups of proteins (COG) difference analysis.

Animals and Treatments
Eighteen platforms were placed in a water tank. Twelve mice were placed in the water bath. Every mouse in the water bath could move from one platform to another by jumping. The water filled the water bath 4 cm from the base. When the mice had reached the rapid eye movement stage of sleep, which is the paradoxical phase of sleep, the mice will fall into the water caused by muscle atonia. The mice then woke and would try to climb up the platform to avoid being drowned. Throughout the experiments, the water was replaced with clean water in the tank.

Cell Culture and Treatment
After 6 h, the medium was changed to basal medium. After 12 h of cultivation, 5 × 10 6 cells/mL and 5 × 10 5 cells/mL were seeded per well in a flat-bottom 96-well culture plate and 12-well culture plate, respectively. Cell proliferation was assessed from the 96-well culture plates using a colorimetric assay based on the reduction of tetrazolium salt (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich). The plates were incubated at 37°C in 5% CO2 for 24 h. Four hours later, 10 μL of MTT was added to the medium (5 mg/mL in complete RPMI-1640 medium). At the end of 28 h, 100 μL of 10% sodium dodecyl sulfate (SDS) was added to each well. The optical density (OD) of each well was measured using a microplate reader (Model 680; Bio-Rad, St. Louis, MO, USA) equipped with a 570 nm wavelength filter. The mean OD values for each triplicate set were used in subsequent statistical analyses. The proliferative activity of IECs was expressed as the stimulation index (SI) as follows: SI = OD570 (stimulated cells)/OD570 (unstimulated cells).

SiRNA Transfection
SiRNA oligonucleotides were designed and produced by Invitrogen (Grand Island, NY).
Sequences (5`-3`) of HDAC3-targeting siRNA were CGGGAUGGCAUUGAUGACCAGAGUU (sense), AACUCUGGUCAUCAAUGCCAUCCCG (antisense); scramble RNA of High GC ratio was used as negative control. The siRNAs were transfected into cells using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions for 24 h before further operations, and the efficacy of silencing was assessed by immunoblotting. Values are presented as the mean ± SE. Differences were assessed by ANOVA and denoted as follows: different lowercase letters: P < 0.05; different uppercase letters: P < 0.01; same letter: P > 0.05.

Figure S2
6 Effect of SD and SR on gut microbiota α diversity and β diversity. PCA (A, D), PCoA (B, E) and NMDS (C, F) score plots based on the Bray-Curtis score plot based on the OTUs for the SD0, SD1, SD2, SR0 and SR groups. Values are presented as the mean ± SE.
Differences were assessed by ANOVA and denoted as follows: different lowercase letters: P < 0.05; different uppercase letters: P < 0.01; same letter: P > 0.05.

Figure S3
Correlation analysis of gut microbiota. 80 significantly changed intestinal microbiota among the three SD groups (A) and two SR groups (B), and these changed 80 intestinal microbiota interacted with each other to mediate intestinal function.

Figure S4
Effect of SD and butyrate supplementation on gut microbiota α diversity.

ACE (A), Chao1 (B), Shannon (C), and Simpson indices (D) of the CON, SD and
SD+Butyrate groups. Values are presented as the mean ± SE. Differences were assessed by ANOVA and denoted as follows: different lowercase letters: P < 0.05; different uppercase letters: P < 0.01; same letter: P > 0.05.